药敏试验M27-A3方案

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1、 940 West Valley Road Suite 1400 Wayne, PA 19087 USA PHONE 610.688.0100 FAX 610.688.0700 E-MAIL: customerserviceclsi.org WEBSITE: www.clsi.org ISBN 1-56238-666-2 (Formerly NCCLS) M27-A3 Vol. 28 No. 14 Replaces M27-A2 Vol. 22 No. 15 Reference Method for Broth Dilution Antifungal Susceptibility Testin

2、g of Yeasts; Approved StandardThird Edition This document addresses the selection and preparation of antifungal agents; implementation and interpretation of test procedures; and quality control requirements for susceptibility testing of yeasts that cause invasive fungal infections. A standard for gl

3、obal application developed through the Clinical and Laboratory Standards Institute consensus process. (Formerly NCCLS) M27-A3 ISBN 1-56238-666-2 Volume 28 Number 14 ISSN 0273-3099 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved StandardThird Edition John H.

4、Rex, MD, FACP Barbara D. Alexander, MD, MHS David Andes, MD Beth Arthington-Skaggs, PhD Steven D. Brown, PhD Vishnu Chaturvedi, PhD Mahmoud A. Ghannoum, MSc, PhD Ana Espinel-Ingroff, PhD Cynthia C. Knapp, MS Luis Ostrosky-Zeichner, MD, FACP Michael A. Pfaller, MD Daniel J. Sheehan, PhD Thomas J. Wal

5、sh, MD Abstract Clinical and Laboratory Standards Institute document M27-A3Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved StandardThird Edition describes a method for testing the susceptibility of antifungal agents to yeast that cause invasive fungal infect

6、ions, including Candida spp. (and Candida glabrata), and Cryptococcus neoformans. Selection and preparation of antifungal agents, implementation and interpretation of test procedures, and the purpose and implementation of quality control procedures are discussed. A careful examination of the respons

7、ibilities of the manufacturer and the user in quality control is also presented. Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved StandardThird Edition. CLSI document M27-A3 (ISBN 1-56238-666-2). Clinical an

8、d Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2008. The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the health care community, is an ongoi

9、ng process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI/NCCLS documents. Current editions are lis

10、ted in the CLSI catalog and posted on our website at www.clsi.org. If your organization is not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax: 610.688.0700; E-Mail: customerserviceclsi.org; Website: www.clsi.org (Formerly NCCL

11、S) Volume 28 M27-A3 v Contents Abstract . . i Committee Membership . iii Foreword . vii Updated Information in This Edition . viii 1 Scope . 1 2 Introduction . 1 3 Standard Precautions . 1 4 Definitions . 1 5 Indications for Performing Susceptibility Tests . 2 6 Antifungal Agents . 3 6.1 Source . 3

12、6.2 Weighing Antifungal Powders . 3 6.3 Preparing Stock Solutions . 4 6.4 Number of Concentrations Tested . 5 6.5 Selection of Antifungal Agents for Routine Testing and Reporting . 5 7 Test Procedures . 6 7.1 Broth Medium . 6 7.2 Preparing Diluted Antifungal Agents . 6 7.3 Inoculum Preparation . 7 7

13、.4 Inoculating RPMI-1640 Medium . 8 7.5 Incubation . 8 7.6 Reading Results . 8 7.7 Interpretation of Results . 8 7.8 Broth Microdilution Modifications . 10 7.9 Trailing Growth and the Impact of Time of Reading . 12 7.10 Other Modifications . 12 8 Quality Control . 12 8.1 Purpose . 12 8.2 QC Responsi

14、bilities . 12 8.3 Selecting Reference Strains . 13 8.4 Storing Reference Strains . 13 8.5 Routine Use of Reference Strains . 14 8.6 Batch of Medium and Lot of Plasticware Control . 15 8.7 QC Frequency . 15 8.8 Other Control Procedures . 16 8.9 QC Strains . 16 References . 17 Appendix A. RPMI 1640 Me

15、dium . 19 Appendix B. McFarland 0.5 Barium Sulfate Turbidity Standard . 20 Summary of Delegate Comments and Committee Responses . 21 The Quality Management System Approach . 24 Related CLSI Reference Materials . 25Volume 28 M27-A3 vii Foreword With the increased incidence of systemic fungal infectio

16、ns and the growing number of antifungal agents, laboratory aids to guide in the selection of antifungal therapy have gained greater attention. In 1982, the CLSI Area Committee for Microbiology formed the Subcommittee on Antifungal Susceptibility Testing. In 1985, this subcommittee published its firs

17、t report 1 in which the results of a questionnaire and a small collaborative study were presented. These results are summarized as follows: Approximately 20% of the responding CLSI membership whose hospitals had greater than 200 beds was performing antifungal testing. Most testing involved broth dil

18、ution methodology. Most strains tested were Candida albicans or other species of yeasts. Most centers tested only a few isolates per year. Agreement in minimal inhibitory concentration (MIC) results among several laboratories that participated in a collaborative study was unacceptably low. Based on

19、these findings, the subcommittee concluded that it would be useful to work toward a more reproducible reference testing procedure. Agreement already existed regarding several elements of the procedure. To facilitate further analysis of various test conditions, the reference method should be a broth

20、macrodilution procedure. Because of examples of drug antagonism by some complex media for certain antifungals, the subcommittee restricted its interest only to fully defined synthetic media. Drug stock solution preparation and dilution procedures previously developed for antibacterial testing proced

21、ures were adopted with minor modifications. Despite agreement in some areas, other factors required additional data to be resolved. These included inoculum preparation; inoculum size; choice among several synthetic media; temperature of incubation; duration of incubation; and end-point definition. T

22、hese factors were the focus of a series of collaborative studies. 2-5 As a result, agreement within the subcommittee was achieved on all of the factors and led to the publication of M27-P in 1992. In the next four years (1992-1996), reference MIC ranges were established for two quality control strai

23、ns for the available antifungal agents, 6,7 and broth microdilution procedures paralleling the broth macrodilution reference procedure became available. 5,8-10 This information was included in a revised standard in 1995 (M27-T). In further revising the document, the subcommittee focused its attentio

24、n on developing relevant breakpoints for available antifungal agents, 11 included in M27-A (1997). Since then, the subcommittee has developed 24- and 48-hour reference MIC ranges for microdilution testing of both established and newly introduced antifungal agents. 12 The results of these studies are

25、 included in the current M27-A3 and M27-S3 (Informational Supplement) 13 documents. Key Words antifungal, broth macrodilution, broth microdilution, susceptibility testing, yeasts Number 14 M27-A3 viii Updated Information in This Edition Definitions (Section 4) Modified definition: Minimal inhibitory

26、 concentration (MIC) Added definition: Antimicrobial susceptibility test interpretive category Quality control Additional Section Indications for performing susceptibility tests (Section 5) Time of reading (Section 7.8.1) Data Inclusion/Exclusion Established numerical scale criteria for visual compa

27、rison of the amount of growth in the control tubes (Section 7.6) Established guide for reading and interpretation of results of Echinocandin antifungals (Sections 7.6.3 and 7.7.8) Expanded recommendations and explanations on acceptable time of reading for antifungal agents when growth is adequate (S

28、ections 7.8.1 and 7.9) Tables All related tables were updated and compiled separately as M27-S3, Informational Supplement instead of a document Appendix. Updates on each table include: Table 1: Interpretive Guidelines for In Vitro Susceptibility Testing of Candida spp. Added new column on “nonsuscep

29、tible (NS)” criteria for interpretive guidelines. Added breakpoints criteria for the following antifungal agents: Anidulafungin Caspofungin Micafungin Voriconazole (first added in M27-S2, published February 2006) Provided additional footnote information for Flucytosine, Anidulafungin, Caspofungin, a

30、nd Micafungin. Table 2: Solvents and Diluents for Preparation of Stock Solutions of Antifungal Agents Added solvents and diluents recommendations for the following antifungal agents: Anidulafungin Caspofungin Micafungin Table 5: Recommended 48-Hour MIC Limits for Two Quality Control and Four Referen

31、ce Strains for Broth Macrodilution Procedures Added information on Issatchenkia orientalis as the known sexual form of Candida krusei. Table 6: Recommended 24- and 48-Hour MIC Limits for Two Quality Control Strains for Broth Microdilution Added the following antifungal agents: Anidulafungin (first a

32、dded in M27-S2, published February 2006) Caspofungin (first added in M27-S2, published February 2006) Micafungin Volume 28 M27-A3 Clinical and Laboratory Standards Institute. All rights reserved. 1 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Y easts; Approved StandardThi

33、rd Edition 1 Scope This document describes a method for testing the susceptibility to antifungal agents of yeasts, including Candida spp. and Cryptococcus neoformans, that cause infections. This method has not been extensively validated for the yeast forms of dimorphic fungi, such as Blastomyces der

34、matitidis or Histoplasma capsulatum variety capsulatum. The subcommittee has focused on developing relevant breakpoints for available antifungal agents, 11 and reference MIC ranges for microdilution testing of both established and newly introduced antifungal agents. 12 Interpretive minimal inhibitor

35、y concentration (MIC) breakpoints and MIC ranges for quality control (QC) isolates are summarized in an Informational Supplement 13 to this document. 2 Introduction The broth macrodilution method described in this document is intended for testing yeasts that cause invasive infections. These yeasts encompass Candida spp. (including Candida glabrata) and C. neoformans. The method has not been used in studies of the yeast forms of dimorphic fungi, such as B. dermatitidis and/or H. capsulatum variety capsulatum. Moreover, testing filamentous fungi (moulds) introduces several additi